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1.  Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry† 
Journal of medicinal chemistry  2010;53(3):1306-1318.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR- and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.
doi:10.1021/jm9015729
PMCID: PMC2836843  PMID: 20085328
2.  Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-D]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter 
The 5-substituted pyrrolo[2,3-d]pyrimidine antifolate pemetrexed (Pmx) is an active agent for malignant pleural mesothelioma (MPM). Pmx is transported into MPM cells by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). We tested the notion that a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 2) might be an effective treatment for MPM, reflecting its highly selective membrane transport by PCFT over RFC. Compound 2 selectively inhibited proliferation of a HeLa subline expressing exclusively PCFT (R1-11-PCFT4) over an isogenic subline expressing only RFC (R1-11-RFC6). By outgrowth, H2452 human MPM cells were highly sensitive to the inhibitory effects of compound 2. By colony-forming assays, following an intermittent (24 h) drug exposure, 2 was cytotoxic. Cytotoxic activity by 2 was due to potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis, as confirmed by nucleoside protection and in situ GARFTase assays with [14C]glycine. Assays with [3H]compound 2 and R1-11-PCFT4 or R1-11-RFC6 cells directly confirmed selective membrane transport by PCFT over RFC. PCFT transport was also confirmed for H2452 cells. In R1-11-PCFT4 and H2452 cells, [3H]compound 2 was metabolized to polyglutamates. Potent in vivo efficacy was confirmed toward early- and upstage H2452 xenografts in severe combined immunodeficient mice administered intravenous compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by novel tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.
doi:10.1007/s00280-013-2094-0
PMCID: PMC3769948  PMID: 23412628
proton-coupled folate transporter; mesothelioma; folate; antifolate; pemetrexed
3.  6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors 
Journal of medicinal chemistry  2015;58(17):6938-6959.
2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]-pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.
doi:10.1021/acs.jmedchem.5b00801
PMCID: PMC4714714  PMID: 26317331
4.  Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry 
Journal of medicinal chemistry  2009;52(9):2940-2951.
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno[2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
doi:10.1021/jm8011323
PMCID: PMC2730022  PMID: 19371039
5.  Synthesis and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier 
Journal of Medicinal Chemistry  2012;55(4):1758-1770.
We reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a 3- (3a) and 4-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells; While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and L-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5 and 6) substitutions on the thienoyl ring, and with acetylenic insertions in the 4-atom bridge, were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.
doi:10.1021/jm201688n
PMCID: PMC3288238  PMID: 22243528
6.  Synthesis and Discovery of High Affinity Folate Receptor-Specific Glycinamide Ribonucleotide Formyltransferase Inhibitors With Antitumor Activity 
Journal of medicinal chemistry  2008;51(16):5052-5063.
A series of 6-substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a 3- to 6-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2, 3, 4 and 5, respectively) was synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the α-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the α-bromoketones, coupling with diethyl-L-glutamate and saponification afforded 2–5. Compounds 2–5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FRα or FRβ, and toward FRα-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogs as antitumor agents.
doi:10.1021/jm8003366
PMCID: PMC2748117  PMID: 18680275
7.  Tumor-Targeting with Novel Non-Benzoyl 6-Substituted Straight Chain Pyrrolo[2,3-d]pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and Inhibition of de novo Purine Nucleotide Biosynthesis 
Journal of medicinal chemistry  2013;56(21):10.1021/jm401139z.
A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a–d with varying chain lengths (n = 5–8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a–d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a–d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a–d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a–d. Hydrolysis and subsequent coupling with diethyl L-glutamate and saponification afforded target compounds 3a–d. Compounds 3b–d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.
doi:10.1021/jm401139z
PMCID: PMC3880613  PMID: 24111942
8.  Novel 5-Substituted Pyrrolo[2,3-d]pyrimidines as Dual Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase and as Potential Antitumor Agents 
Journal of medicinal chemistry  2015;58(3):1479-1493.
A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6–11 with varying chain lengths (n = 1–6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6–11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8–10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure–activity relationship for transport and dual target inhibition.
doi:10.1021/jm501787c
PMCID: PMC4714715  PMID: 25602637
9.  Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine antifolates as dual acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: implications of inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase to AMPK activation and anti-tumor activity 
Journal of medicinal chemistry  2013;56(24):10016-10032.
We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5–10) with 1 to 6 bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analog and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Anti-proliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.
doi:10.1021/jm401328u
PMCID: PMC3917155  PMID: 24256410
10.  The human proton-coupled folate transporter 
Cancer Biology & Therapy  2012;13(14):1355-1373.
This review summarizes the biology of the proton-coupled folate transporter (PCFT). PCFT was identified in 2006 as the primary transporter for intestinal absorption of dietary folates, as mutations in PCFT are causal in hereditary folate malabsorption (HFM) syndrome. Since 2006, there have been major advances in understanding the mechanistic roles of critical amino acids and/or domains in the PCFT protein, many of which were identified as mutated in HFM patients, and in characterizing transcriptional control of the human PCFT gene. With the recognition that PCFT is abundantly expressed in human tumors and is active at pHs characterizing the tumor microenvironment, attention turned to exploiting PCFT for delivering novel cytotoxic antifolates for solid tumors. The finding that pemetrexed is an excellent PCFT substrate explains its demonstrated clinical efficacy for mesothelioma and non-small cell lung cancer, and prompted development of more PCFT-selective tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that derive their cytotoxic effects by targeting de novo purine nucleotide biosynthesis.
doi:10.4161/cbt.22020
PMCID: PMC3542225  PMID: 22954694
folate; antifolate; transport; proton-coupled folate transporter; reduced folate carrier; tumor microenvironment
11.  Synthesis and evaluation of a classical 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates☆ 
Bioorganic & medicinal chemistry  2006;14(24):10.1016/j.bmc.2006.08.029.
Two classical antifolates, a 2,4-diamino-5-substituted furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine, were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The syntheses were accomplished by condensation of 2,6-diamino-3(H)-4-oxo-pyrimidine with α-chloro-ketone 21 to afford two key intermediates 23 and 24, followed by hydrolysis, coupling with l-glutamate diethyl ester and saponification of the diethyl ester to afford the classical antifolates 13 and 14. Compounds 13 and 14 with a single carbon atom bridge are both substrates for folylpoly-γ-glutamate synthetase (FPGS), the enzyme responsible for forming critical poly-γ-glutamate antifolate metabolites with increased potency and/or increased cell retention. Compound 14 is a highly efficient FPGS substrate demonstrating that 2,4-diamino-5-substituted furo[2,3-d]pyrimidines are important lead structures for the design of antifolates with FPGS substrate activity. It retains inhibitory potency for DHFR and TS compared to the two atom bridged analog 5. Compound 13 is a poor inhibitor of purified DHFR and TS, and both 13 and 14 are poor inhibitors of the growth of CCRF-CEM human leukemia cells in culture, indicating that single carbon bridged compounds in these series though conducive to FPGS substrate activity were not potent inhibitors.
doi:10.1016/j.bmc.2006.08.029
PMCID: PMC3850758  PMID: 16990006
Pyrrolo[2,3-d]pyrimidines; Antifolates; Dihydrofolate reductase
12.  Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells. 
British Journal of Cancer  1998;78 (Suppl 3):27-34.
LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.
PMCID: PMC2062799  PMID: 9717988
13.  Biology of the Major Facilitative Folate Transporters SLC19A1 and SLC46A1 
Current topics in membranes  2014;73:175-204.
This chapter focuses on the biology of the major facilitative membrane folate transporters, the reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT). Folates are essential vitamins, and folate deficiency contributes to a variety of heath disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates intestinal absorption of dietary folates. Clinically relevant antifolates such as methotrexate (MTX) are transported by RFC, and the loss of RFC transport is an important mechanism of MTX resistance. PCFT is abundantly expressed in human tumors and is active under pH conditions associated with the tumor microenvironment. Pemetrexed (PMX) is an excellent substrate for PCFT as well as for RFC. Novel tumor-targeted antifolates related to PMX with selective membrane transport by PCFT over RFC are being developed. The molecular picture of RFC and PCFT continues to evolve relating to membrane topology, N-glycosylation, energetics, and identification of structurally and functionally important domains and amino acids. The molecular bases for MTX resistance associated with loss of RFC function, and for the rare autosomal recessive condition, hereditary folate malabsorption (HFM), attributable to mutant PCFT, have been established. From structural homologies to the bacterial transporters GlpT and LacY, homology models were developed for RFC and PCFT, enabling new mechanistic insights and experimentally testable hypotheses. RFC and PCFT exist as homo-oligomers, and evidence suggests that homo-oligomerization of RFC and PCFT monomeric proteins may be important for intracellular trafficking and/or transport function. Better understanding of the structure and function of RFC and PCFT should facilitate the rational development of new therapeutic strategies for cancer as well as for HFM.
doi:10.1016/B978-0-12-800223-0.00004-9
PMCID: PMC4185403  PMID: 24745983
14.  The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared to the Reduced Folate Carrier 
Molecular pharmacology  2008;74(3):854-862.
The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This paper addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, ZD1694 or PT523 in comparison to RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed with an influx Km of 0.2 – 0.8 µM at pH 5.5 in these systems. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed due to impaired transport because of the redundancy of these genetically distinct routes.
doi:10.1124/mol.108.045443
PMCID: PMC2716086  PMID: 18524888
15.  Regulation of proton-coupled folate transporter in retinal Müller cells by the anti-psoriatic drug monomethylfumarate 
Glia  2011;60(3):333-342.
Fumaric acid esters are used to treat psoriasis, an inflammatory skin disease characterized by keratinocyte proliferation. Inflammation and proliferation are hallmarks of retinal disease; hence, fumaric acid esters may have therapeutic value in retinal pathology. In diseased retinas, Müller glial cells (MCs) undergo reactive gliosis, a hyperproliferative state. MCs take up folate, a vitamin necessary for cell proliferation, via the proton-coupled folate transporter (PCFT). Here we examined the effect of monomethylfumarate (MMF), the active metabolite of fumaric acid esters, on expression and function of PCFT in MCs. Primary MCs, isolated from neonatal mouse retinas, were treated with MMF, and PCFT function was monitored by measuring uptake of radiolabeled methyltetrahydrofolate (MTF) at pH 5.5. Dose-response and time-course analyses were performed to identify optimal conditions for maximal effect. The influence of MMF treatment on kinetic parameters of PCFT was studied, and PCFT expression was analyzed at the mRNA and protein level. MTF uptake in MCs decreased by ~50% following 18 h treatment with 1 mM MMF. This effect was specific to fumaric acid esters. MMF treatment decreased the maximal velocity of the transporter without altering substrate affinity. The decrease in PCFT function following MMF treatment was accompanied by attenuated PCFT expression. This is the first report that an antipsoriatic compound can regulate folate transport in MCs and may have potential for the treatment of reactive gliosis in retinal disease.
doi:10.1002/glia.22266
PMCID: PMC3265662  PMID: 22072423
fumaric acid esters; vitamin transport; folic acid; Müller cell gliosis; mouse
16.  Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6 
The Biochemical journal  2015;469(1):33-44.
The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.
doi:10.1042/BJ20150169
PMCID: PMC4713120  PMID: 25877470
anti-folate; cross-linking; folate; oligomerization; proton-coupled folate transporter; transporter
17.  Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method† 
Biochemistry  2010;49(13):2925-2931.
The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption. Loss-of-function mutations in this gene are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption. In this study, the substituted cysteine accessibility method was utilized to localize extra- or intra-cellular loops connecting predicted PCFT transmembrane domains. Cysteine-less PCFT was generated by replacement of all seven cysteine residues with serine, and was shown to be functional, following which cysteine residues were introduced into predicted loops. HeLa cells, transiently transfected with these PCFT mutants, were then labeled with an impermeant, cysteine-specific biotinylation reagent (MTSEA-biotin) with or without permeabilization of cells. The biotinylated proteins were precipitated by streptavidin beads and assessed by Western blotting analysis. The biotinylation of PCFT was further confirmed by blocking cysteine residues with impermeant 2-sulfonatoethyl methanethiosulfonate. Two extracellular cysteine residues (66, 298) present in WT-PCFT were not biotinylated; however, in the absence of either one, biotinylation occurred. Likewise, biotinylation occurred after treatment with β-mercaptoethanol. Taken together, these analyses establish a PCFT secondary structure of twelve transmembrane domains with the N- and C- termini directed to the cytoplasm. The data indicate further that there is a disulfide bridge, which is not required for function, between the native C66 and C298 residues in the first and fourth transmembrane domains, respectively.
doi:10.1021/bi9021439
PMCID: PMC2866095  PMID: 20225891
18.  HYPERMETHYLATION OF THE HUMAN PROTON-COUPLED FOLATE TRANSPORTER (PCFT, SLC46A1) MINIMAL TRANCRIPTIONAL REGULATORY REGION IN AN ANTIFOLATE-RESISTANT HELA CELL LINE 
Molecular cancer therapeutics  2009;8(8):2424-2431.
This laboratory recently identified a novel proton-coupled folate transporter (PCFT) that mediates intestinal folate absorption and transport of folates into the central nervous system. The present study focuses on the definition of the minimum transcriptional regulatory region of this gene in HeLa cells and the mechanism(s) underlying the loss of PCFT expression in the methotrexate-resistant HeLa R1–11 cell line. The PCFT transcriptional regulatory controls were localized between −42 and +96 bases from the transcriptional start site using a luciferase-reporter gene system. The promoter is a G+C rich region of 139 nucleotides contained in a CpG island. HeLa R1–11 cells have no mutations in the PCFT open-reading-frame and its promoter; the transcription/translation machinery is intact since transient transfections in HeLa R1–11 and wild-type HeLa cells produced similar luciferase activities. Hypermethylation at CpG sites within the minimal transcriptional regulatory region was demonstrated in HeLa R1–11 cells as compared to the parental PCFT-competent HeLa cells, using bisulfite conversion and sequence analysis. Treatment with 5-aza-2’-deoxycytidine resulted in a substantial restoration of transport and PCFT mRNA expression and small, but significant decreases in methylation in the promoter region. In vitro methylation of the transfected reporter plasmid inhibited luciferase gene expression. Cytogenetics/FISH indicated a loss of half the PCFT gene copies in HeLa R1–11 as compared to PCFT-competent HeLa cells. Taken together, promoter silencing via methylation and gene copy loss accounted for the loss of PCFT activity in antifolate-resistant HeLa R1–11 cells.
doi:10.1158/1535-7163.MCT-08-0938
PMCID: PMC2735101  PMID: 19671745
PCFT; HCP1; proton-coupled folate transporter; PCFT regulation; epigenetic regulation; antifolate-resistance; methotrexate; pemetrexed
19.  Synthesis and Biological Activity of N4-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as Vascular Endothelial Growth Factor Receptor-2 Inhibitors and Antiangiogenic and Antitumor Agents 
Bioorganic & medicinal chemistry  2010;18(10):3575-3587.
A series of eight N4-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8–15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N4-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16–20 were synthesized to evaluate the importance of the 2-NH2 moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of α-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of α-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6- phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40–42 respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8–20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.
doi:10.1016/j.bmc.2010.03.052
PMCID: PMC2868963  PMID: 20403700
Pyrrolo[2,3-d]pyrimidines; Receptor tyrosine kinase inhibitors; Antiangiogenic agents; Antitumor agents
20.  The Effect of 5-Alkyl Modification on the Biological Activity of Pyrrolo[2,3-d]pyrimidine Containing Classical and Nonclassical Antifolates as Inhibitors of Dihydrofolate Reductase and as Antitumor and/or Antiopportunistic Infection Agents1a-e 
Journal of medicinal chemistry  2008;51(15):10.1021/jm800244v.
Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of α-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-l-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 ≤ 10−7 M. Nonclassical 17 (IC50 = 58 nM) was a potent inhibitor of Toxoplasma gondii (T. gondii) DHFR with >500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.
doi:10.1021/jm800244v
PMCID: PMC3858179  PMID: 18605720
21.  Cloning and Functional Characterization of the Proton-coupled Electrogenic Folate Transporter and Analysis of its Expression in Retinal Cell Types 
Investigative ophthalmology & visual science  2007;48(11):10.1167/iovs.07-0288.
PURPOSE
We have previously investigated the cellular uptake of folate in the retina. Recently, a new proton-coupled folate transporter (PCFT) in human intestine was reported. Here we investigated the expression of this novel transporter in the retina, cloned the mouse ortholog from retinal tissue, and characterized its transport function.
METHODS
RT-PCR and folate uptake measurements were used to detect the expression of PCFT in mouse retina and in retinal cell types. Expression of PCFT mRNA in intact retina was investigated by in situ hybridization. Mouse PCFT cDNA was cloned and its transport characteristics were analyzed by electrophysiological methods following expression of the cloned transporter in X. laevis oocytes.
RESULTS
RT-PCR showed expression of PCFT mRNA in both neural retina and RPE-eyecup. In situ hybridization detected PCFT mRNA in all retinal cell layers. Proton-coupled folate uptake was detectable in primary cultures of ganglion, Müller, and RPE cells of mouse retina, and in RPE, ganglion, and Müller cell lines of human or rat origin. In X. laevis oocytes expressing the cloned mouse PCFT, folate and its derivatives methotrexate and 5-methyltetrahydrofolate induced H+-coupled inward currents with Kt values of 1.2 ± 0.1, 4.6 ± 0.5 and 3.5 ± 0.8 µM, respectively. The transport process showed a H+:folate stoichiometry of 1:1, suggesting that PCFT transports the zwitterionic form of folate.
CONCLUSIONS
This is the first report on the expression of PCFT in the retina. All cell layers of the retina express this transporter. Mouse PCFT, cloned from retina, mediates H+-coupled electrogenic transport of folate and its derivatives.
doi:10.1167/iovs.07-0288
PMCID: PMC3850295  PMID: 17962486
22.  Membrane Transporters and Folate Homeostasis; Intestinal Absorption, Transport into Systemic Compartments and Tissues 
Folates, the generic term for the family of B vitamins, are derived entirely from dietary sources, and are key one-carbon donors required for de novo nucleotide and methionine synthesis. These highly hydrophilic molecules utilize genetically distinct and functionally diverse transport systems to enter cells: the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT), and the folate receptors. Each plays a unique role in mediating folate transport across epithelia and into systemic tissues. With the recent discovery of the mechanism of intestinal folate absorption, and the clarification of the genetic basis for the autosomal recessive disorder, hereditary folate malabsorption, involving loss-of-function mutations in PCFT protein, it is now possible to piece together how these folate transporters contribute, both individually and collectively, to folate homeostasis in humans. This review focuses on the physiological roles of these major folate transporters with a brief consideration of their impact on the pharmacological activities of antifolates.
doi:10.1017/S1462399409000969
PMCID: PMC3770294  PMID: 19173758
23.  The proton-coupled folate transporter: physiological and pharmacological roles 
Current opinion in pharmacology  2013;13(6):875-880.
Summary
Recent studies have identified the proton-coupled folate transporter (PCFT) as the mechanism by which folates are absorbed across the apical brush-border membrane of the small intestine and across the basolateral membrane of the choroid plexus into the cerebrospinal fluid. Both processes are defective when there are loss-of-function mutations in this gene as occurs in the autosomal recessive disorder hereditary folate malabsorption. Because this transporter functions optimally at low pH, antifolates are being developed that are highly specific for PCFT in order to achieve selective delivery to malignant cells within the acidic environment of solid tumors. PCFT has a spectrum of affinities for folates and antifolates that narrows and increases at low pH. Residues have been identified that play a role in folate and proton binding, proton coupling, and oscillation of the carrier between its conformational states.
PMCID: PMC4100332  PMID: 24383099
24.  The membrane transport and polyglutamation of pralatrexate, a new-generation dihydrofolate reductase inhibitor 
Purpose
To characterize, directly and for the first time, the membrane transport and metabolism of pralatrexate, a new-generation dihydrofolate reductase inhibitor approved for the treatment of peripheral T-cell lymphoma.
Experimental Design
[3H]pralatrexate transport was studied in unique HeLa cell lines that express either the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT). Metabolism to active polyglutamate derivatives was assessed by liquid chromatography. These properties were compared to those of methotrexate (MTX).
Results
The pralatrexate influx Kt, mediated by RFC, the major route of folate/antifolate transport at systemic pH, was 0.52 , 1/10th the MTX influx Ki. The electrochemical-potential of pralatrexate within HeLa cells far exceeded the extracellular level and was greater than for MTX. In contrast, MTX transport mediated by PCFT, the mechanism of folate/antifolate absorption in the small intestine, exceeded that for pralatrexate. After a 6h exposure of HeLa cells to 0.5 μM pralatrexate, 80% of intracellular drug was its active polyglutamate forms, predominantly the tetraglutamate, and was suppressed when cells were loaded with natural folates. There was negligible formation of MTX polyglutamates. The difference in pralatrexate and MTX growth inhibition was far greater after transient exposures (375-fold) than continuous exposure (25-fold) to the drugs.
Conclusion
Pralatrexate’s enhanced activity relative to MTX is due to its much more rapid rate of transport and polyglutamation, the former less important when the carrier is saturated. The low affinity of pralatrexate for PCFT predicts a lower level of enterohepatic circulation, and increased fecal excretion of the drug relative to MTX.
doi:10.1007/s00280-013-2231-9
PMCID: PMC3784351  PMID: 23881211
Methotrexate; PCFT; polyglutamation; pralatrexate; RFC
25.  Substituted Pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis Thymidylate Synthase Inhibitors 
Bioorganic & medicinal chemistry letters  2013;23(19):10.1016/j.bmcl.2013.07.037.
Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals1, 2. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2’-chlorophenyl with a sulfur bridge with a Ki of 8.83 ± 0.67 nM is discussed in terms of several Van de Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors.
doi:10.1016/j.bmcl.2013.07.037
PMCID: PMC3853131  PMID: 23927969
Pyrrolo[2,3-d]pyrimidines; Thymidylate synthase; Dihydrofolate reductase; Cryptospridium hominis

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